化学
碎片(计算)
劈理(地质)
乙二胺四乙酸
组氨酸
特里斯
单克隆抗体
免疫球蛋白轻链
色谱法
螯合作用
生物化学
抗体
氨基酸
有机化学
岩土工程
免疫学
工程类
操作系统
生物
断裂(地质)
计算机科学
作者
Yilue Zhang,Christian Schöneich
标识
DOI:10.1021/acs.molpharmaceut.2c00840
摘要
Fragmentation of therapeutic monoclonal antibodies represents a critical quality attribute. Here, we report a novel visible light-induced heavy chain fragmentation of IgG1 mediated by an Fe(III)-containing histidine (His) buffer. Based on non-reducing sodium dodecylsulfate-polyacrylamide gel electrophoresis and mass spectrometry analysis, IgG1 fragments with apparent molecular weights of ∼130, ∼110, and ∼22 kDa were detected in photo-irradiated samples and were mechanistically rationalized with an oxidative cleavage at Thr259. Specifically, the reactions are proposed to involve the generation of an intermediary alkoxyl radical, which undergoes β-cleavage to yield a glycyl radical. The latter either converts into Gly or adds oxygen and follows a peroxyl radical chemistry. The cleavage process requires the presence of His, while only negligible yields of cleavage products are formed when His is replaced by acetate, succinate, or phosphate buffer. Importantly, the fragmentation can be prevented by ethylenediaminetetraacetic acid (EDTA) only when the EDTA concentrations are in significant excess over the concentrations of Fe(III) and proteins, suggesting a strong binding between Fe(III) and IgG1.
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